用唾液代替外周血检测丙型肝炎抗体的探讨

目的：探讨口腔唾液代替外周血标本检测丙型肝炎病毒(Hepatitis C Virus，HCV)抗体的可行性。方法：用ELISA法稍加改进，分别对180例患者外周血和唾液标本中的HCV抗体(抗-HCV)进行平行检测，地比分析。结果：180例患者中，外周血抗—HCV62例阳性，唾液标本抗-HCV60例阳性，阴性2例；外周血抗-HCV118例阴性，唾液抗—HCV也是118例阴性。与外周血相比，唾液标本的灵敏度为96．8％(60／62)特异性为100％(118／118)。结论：唾液抗-HCV检测可用于HCV感染的临床诊断及流行病学调查。     

应用丙型肝炎病毒蛋白多区段合成肽检测特异性抗体的探讨

应用丙型肝炎病毒(HCV)的核心(C)区和非结构(NS)区4条合成肽(CP_(10)、CP_9、CP_(33C)和CP_(100)),建立了ELISA检测抗-HCV的方法,并用逆转录聚合酶链反应(RT-PCR)检测血清HCV RNA作对比研究。发现在检测来自肝炎患者、供血员和其它疾病患者的320份血清标本中,有49份血清对1条或多条合成肽呈阳性反应;单一合成肽抗体检出率以CP_(10)最高(9.69％),CP_(33C)最底(3.44％),联合应用C区和NS区合成肽可显著提高抗体的检出率(P<0.01);57.14％～88.24％抗体阳性血清用RT-PCR可检出HCV RNA。提示应用合成肽检测本地区HCV感染者特异性抗体是可行的,联合应用多区段合成肽对减少假阴性的发生有重要意义。     

部分抗-HCV阴性和阳性血清丙肝抗体分片段检测分析

目前,ELISA抗-HCV作为血液(HCV)传染指标的主要检测手段,其检测结果的可靠性至关重要。我们对部分ELISA抗-HCV阴性和阳性的血清进行丙肝抗体分片段检测,以探讨ELISA抗-HCV不同区域抗体的反应性和血液HCV感染的真实情况,现将结果报告如下。     

255例抗-HCV与HCV RNA定量检测结果比较分析

目的 探讨血清丙型肝炎病毒抗体和核糖核酸检测阳性结果的临床意义。方法 采用第三代试剂药盒ELISA法检测抗-HCV、荧光定量聚合酶链反应(FQ-PCR)法检测HCV RNA。结果 抗-HCV阳性／HCV RNA阴性135例(52．94％),抗-HCV／HCV RNA均阳性者99例(38．82％),而抗-HCV阴性／HCV RNA阳性者21例(8．24％)。结论 临床上疑诊丙型肝炎病毒感染的患者在检测抗-HCV的同时,应加做HCV RNA定量检测。     

无偿献血者HCV－CAg检测的可行性探讨

目的：探讨在无偿献血者中开展丙型肝炎病毒核心抗原(HCV-CAg)检测的可能性及意义。方法：采用美国强生(ORTH0)公司HCV核心抗原酶联免疫(ELISA)检测试剂，对无偿献血者血样进行检测，并对结果进行分析，阳性者用荧光定量PCR方法进行HCV检测，并将抗体检测、抗原检测、NAT检测的结果进行比较。结果：献血者合格血样903份(抗-HCV检测阴性)中未发现HCVC抗原阳性者，不合格血样(n=197)共发现6份阳性(6／197)：HBsAg( )中有l份阳性(1／43)，抗-HCV( )中有4份阳性(4／56)，ALT( )中有1份阳性(1／128)；抗-HIv( )12、抗-PT( )3中未检出阳性。结论：现行的抗-HCV检测方法有可能出现HCV感染的漏检，HCV C抗原检测方法可提高检测的灵敏度，在献血者中开展该项检测是可行的。     

HCV感染的不同临床型HCV核心非结构区抗体检测结果和稳定性研究

目的:研究HCV感染的不同临床型HCV核心区、非结构区抗体检测结果及其稳定性。方法:采用酶联免疫试验(EIA)检测不同功能区抗体，聚合酶链反应(PCR)测定其逆转录病毒。结果C区C11与C22抗体检测结果各临床型均具良好的一致性；NS3区C7区与C33抗体检测结果差异较大。单片段C区C11抗体与Ns3区抗体C7抗体具有早期检测的价值，各临床型反应性较强(S／CO值较高)；且二者互补性较强，尤其HCV相关肝癌。HCV抗体检测不能见出所有HCV感染者，抗HCV阳性者可部分或大部分RT—PCR阳性；RT—PCR能检出HCV阴性标本，且均为急性感染和无症状ALT正常者。HCV相关肝癌HCV抗体和RT—PCR反应性较低。单片段C11抗体极易缺失或(和)变异，阳性者均出现病毒血症中；NS3抗体单独阳性多见，却少与病毒血症共存；NS4．5抗体亦可单独阳性。结论:C、NS3抗体出现较早，反应性强，检出率高，具有早期诊断价值。NS4、Ns5抗体出现较晚，在慢性感染时检出率较高，HCV抗体检测加入NS4、NS5抗体可提高其检测水平。RT—PCR对确诊HCV感染具有重要意义。     

HCV核心抗原在丙肝检测中的应用分析

目的：探讨丙型肝炎病毒（HCV）核心抗原在丙肝检测中的应用效果。方法选取100份HCV感染患者血清标本作为研究对象,所有标本经丙型肝炎病毒核糖核酸（HCV-RNA）检测均为阳性,给予HCV、HCV-cAg检测,比较检测结果符合率。结果 HCV核心抗原测定符合率为93.0%,抗-HCV抗体检测符合率为81.0%,HCV核心抗原测定法符合率明显高于抗-HCV抗体检测法,组间比较差异显著（P<0.05）。结论 HCV核心抗原检测丙肝具有更高的检出率,应用价值较高,且操作便捷、成本低,值得推广应用。     

丙型肝炎病毒检测与抗体检测的比较

应用ELISA法检测抗-HCV抗体的阳性检出率不及PCR法检测HCV RNA。据文献报道HCV的感染率高于抗-HCV抗体检测的结果,而PCR法可检出携带者血内少量的丙型肝炎病毒,因此被视为较有效的检测手段。我们对本院各科送检丙型肝炎的部分标本及少量献血员标本进行抗-HCV抗体和HCVRNA的检测,以比较两种方法在临床检验中的意义。     

酶联免疫吸附试验检测丙型肝炎病毒核心抗原的临床价值

目的探讨酶联免疫吸附试验（ELISA）法检测丙型肝炎核心抗原（HCV—Ag）诊断丙型肝炎病毒（HCV）感染的方法及临床价值。方法采用ELISA法检测疑为HCV感染患者86例,采血半小时内分离出血浆,部分于当日进行丙型肝炎病毒核糖核酸（HCV—RNA）和丙型肝炎病毒抗体（HCV—Ab）检测,其余置-20℃冰箱保存,用于HCV—Ag检测。结果HCV—IqNAPCR、HCV-Ag、抗-HCV阳性率分别为46．5l％、43．02％、37．21％;同HCV～RNAPCR检测结果显示,HCV—Ag检测符合率89．53％（77／86）、假阳性率3．49％（3／86）、假阴性率6．98％（6／86）;抗-HCV检测符合率81．40％（70／86）、假阳性率4．65％（4／86）、假阴性率13．95％（12／86）。结论ELISA法检测HCV—Ag敏感性及特异性较高,可明显缩短HCV感染的窗口期,且简捷、价廉,适合大规模筛查和基层医院开展。     

HCV-RNA检测在提高HCV感染患者检出率的试验研究

目的:探讨组合检测丙肝抗体和丙肝核心抗原并联合丙肝RNA在丙肝临床诊断中的意义。方法:对13 117例患者进行HCV抗体和HCV抗原组合检测,对两种方法中的阳性标本进行HCV-RNA确证检测。结果:13 117例患者中,丙肝抗体阳性188例,丙肝核心抗原阳性52例,其中丙肝抗体和核心抗原均阳性者48例,单独核心抗原阳性4例,总阳性数192例。丙肝抗体阳性188例中HCV抗体阳性标本中有121例HCV-RNA阳性,检出率为64.4%;有48例HCV-c Ag阳性,检出率为25.5%(48/188)。丙肝抗体和核心抗原均阳性者HCVRNA阳性45例,检出率为93.75%;单独核心抗原阳性4例中有3例HCV-RNA阳性,检出率75%。结论:抗-HCV和HCV-c Ag组合作为HCV感染初筛实验联合HCV-RNA确证检测,对HCV感染患者阳性检出率更高,假阳性率更低,可作为临床对HCV感染患者早期诊断和治疗的有效检验程序。     

Clinical application and analysis of hepatitis C virus NS3 antigen detection by ELISA in human serum

Background Hepatitis C virus （HCV） core antigen assays have been produced to exclude infectious donations collected during the preseroconversion window phase （PWP）. For the same purpose, we evaluated the specificity and sensitivity of a novel hepatitis C virus NS3 antigen detection immunoassay and the application of this assay in clinical diagnosis. Methods Samples from 77 healthy subjects, 173 anti-HCV positive patients and 3708 hepatitis patients other than HCV positive were tested with the HCV NS3 antigen assay. Some HCV NS3 antigen positive samples were further validated with HCV-RNA, neutralization and immunodot assays. Twenty-five sequential samples from 11 HCV NS3 antigen positive patients were subjected to kinetic study. Results Only 48 （1.3%） of 3708 anti-HCV negative samples were positive for HCV NS3 antigen. Among them, 44 of 3030 samples from patients only infected with HBV were HCV NS3 antigen positive, 4 of the 445 samples from patients infected with other type hepatitis were HCV NS3 antigen positive. In addition, 42 （24.3%） of 173 anti-HCV positive samples were HCV NS3 antigen positive and all 77 samples from healthy subjects were negative to HCV NS3 antigen assay. Of the 15 HCV NS3 antigen positive samples, 9 （60%） were HCV-RNA positive. The neutralization and positive percentage of immunodot assay for 23 HCV NS3 antigen positive sera were 87.0% （20/23） and 69.6% （16/23） respectively. Of the 25 sequential samples from 11 HCV NS3 antigen positive patients, there was a negative correlation between the OD values and the duration of test （r=-0.989, P〈0.05）, and there were correlations among their HCV NS3 antigen, HCV-RNA and anti-HCV Utres. The anti-HCV antibodies of two sera were detected while their OD values of HCV NS3 antigen decreased gradually. Conclusions The HCV NS3 antigen detection assay showed perfect specificity and high sensitivity. Thus, it would be useful and economical as a routine test in laboratories for early diagnosis of HCV infection and prevention.     

荧光定量巢式逆转录PCR(FQ-nRT-PCR)在检测HCV-RNA中的意义

目的:采用荧光定量巢式逆转录聚合酶链反应(FQ-nRT-PCR)检测丙型肝炎患者外周血清HCV-RNA,探讨HCV-RNA的临床应用价值。方法:用FQ-nRT-PCR和酶联免疫吸附试验(ELISA)分别检测192例HCV感染患者血清,比较HCV-RNA和抗-HCV的临床意义。结果:192例丙型肝炎患者血清标本中,HCV-RNA检出率为75.53%(145/192),抗-HCV检出率为91.66%(176/192)。急性丙型肝炎患者HCV-RNA定量均值达5.21×106拷贝/ml,慢性肝炎次之,肝硬化相对偏低,为5.14×103拷贝/ml。结论:192例丙型肝炎患者抗-HCV检出率高于HCV-RNA检出率,但HCV-RNA对丙型肝炎的早期诊断具有灵敏度高、定量准、假阳性率低等优点,此外在病毒复制水平及抗病毒治疗的疗效评估方面具有重要的临床价值。     

High prevalence of hepatitis C virus-ribonucleic acid positivity in anti-hepatitis C virus negative renal transplant patients

Background

Hepatitis C virus (HCV) infection is common in renal transplant (RT) patients. Some of these patients remain anti-HCV negative despite presence of infection and these are identified by a positive HCV-ribonucleic acid (RNA) test.

Methods

We studied 404 RT patients for prevalence of HCV-RNA positivity in anti-HCV negative patients. Serum was tested for presence of anti-HCV antibodies using a third generation HCV micro-ELISA (enzyme-linked immunosorbent assay) test, which utilises a combination of HCV structural and nonstructural antigens. The RNA was extracted from patient serum for HCV viral quantification using Quiagen Ql Amp Viral RNA mini extraction kit. The HCV-RNA viral load was performed on Corbet Rotor Gene 3000 thermocycler using Taqman principle.

Results

About 308 patients were anti-HCV negative and 96 were anti-HCV positive, resulting in prevalence of overt HCV infection of 23.7%. A total of 130 anti-HCV negative patients tested positive for HCV-RNA making a prevalence of occult HCV infection of 42.2%. There was no significant difference in the rate of overt or occult HCV infection between males and females. Patients with HCV infection (whether overt or occult) had received more number of dialysis sessions (62.5 vs 32.2) and blood transfusions (2.78 vs 1.99) when compared to those without HCV infection (P=0.001). The mean duration on dialysis was also longer (8.15 months vs 4.53 months) in patients with HCV infection (P= 0.0001).

Conclusion

A direct test for HCV viraemia is important to accurately determine the epidemiology of HCV infection in RT patients who remain anti-HCV negative despite harbouring active HCV infection.Key Words: hepatitis C, real time PCR, renal replacement therapy     

Hepatitis C virus infection in different groups of children in Wuhan area

Summary To investigate the incidence of child’s HCV infection in our area, 637 children with different background, including 65 posttransfusion cases, 419 hepatitis patients (250 cases of acute hepatitis A, 156 cases of chronic hepatitis B and 13 cases of non-A, non-B hepatitis), 50 infantile hepatitis syndrome (1HS) infants and 103 healthy day-cared children were tested for serum anti-HCV antibody (EIA) and HCV RNA (nested PCR). It was found that posttransfusion children had significantly higher anti-HCV positive rate (30. 8%) and HCV infection incidence (43.1%) than hepatitis patients (4.3% and 5.3%), IHS infants (6.0% and 8.0%) and daycared children (2.9% and 2.9%). 25 of 33 cases with posttransfusion hepatitis (PTH) developed hepatitis C, which was the leading cause of PTH (75.8%) and NANB PTH (25/30, 83.3%). The incidence of HCV infection in NANBH patients was 23.1% (3/13) which was apparently higher than that in day-cared children (P <0. 02) and lower than that in PTH patients (P<0. 001), but not statistically different from that in AHA and CHB patients (P>0. 05). Mother-infant paired study in IHS group showed that 4 pairs of mother-infant had HCV infection, one boy aged 8 months and his mother were anti-HCV positive, and another 3 pairs possessed HCV RNA in sera. 3 of 103 healthy day-cared children were found to have inapparent HCV infection, who were anti-HCV and HCV RNA positive.     

丙型肝炎病毒不同编码区His融合抗原的表达与纯化

目的:表达和纯化丙型肝炎病毒(HCV)-Core、NS3和NS4的His融合抗原H-C、H-NS3和H-NS4,并初步探讨其在抗体检测中的应用。方法:用重组基因工程技术,构建3种重组质粒C-pET-28a-c( )、NS3-pET-28a-c( )和NS4-pET-28a-c( )以及相应的工程菌;质粒经双酶切、PCR和测序鉴定正确后,IPTG诱导抗原表达,从IPTG使用浓度、诱导温度与时间3个方面优化抗原表达条件;用NTA柱纯化抗原后,分别用单片段重组抗原和混合抗原以酶联免疫吸附试验(ELISA)检测100份HCV抗体阳性血清和40份健康人血清。结果:用单片段重组抗原和混合抗原检测的100份HCV抗体阳性血清中,H-C、H-NS3和H-NS4的抗体阳性率分别为91%、75%和52%,而H-C、H-NS3和H-NS4混合抗原的抗体检出率为100%;检测40份健康人血清,结果全部为阴性。结论:HCV不同编码区单片段His融合抗原具有不同的抗体检出率,但均低于混合抗原的阳性率;在开发HCV抗体诊断试剂时,应采用HCV混合抗原。     

献血员血清抗－HCV－IgM检测及其意义

目的探讨献血员血清抗－ＨＣＶ－ＩｇＭ与ＨＣＶ感染的关联性。方法献血员血清８３６份。以ＨＣＶ混合抗原包被聚苯乙烯反应板微孔，以辣根过氧化物酶标记的羊抗人ＩｇＭ单克隆抗体为酶标抗体，建立ＥＬＩＳＡ检测血清抗－ＨＣＶ－ＩｇＭ。以第二代ＥＬＩＳＡ法检测抗－ＨＣＶ－ＩｇＧ，以ＰＣＲ检测血清抗－ＨＣＶ－ＩｇＧ或抗－ＨＣＶ－ＩｇＭ阳性样品中的ＨＣＶ－ＲＮＡ。结果２－巯基乙醇破坏试验及ＨＣＶ抗原抑制试验结果显示，以所建立的ＥＬＩＡＳ检测到的为ＨＣＶ特异ＩｇＭ，批内变异系数为３．４７％，批间变异系数为７．０６％。８３６份献血员血清中，有２份抗－ＨＣＶ－ＩｇＧ阴性而抗－ＨＣＶ－ＩｇＭ和ＨＣＶ－ＲＮＡ均阳性。结论本研究中建立的ＥＬＩＳＡ可较好地用于检测血清抗－ＨＣＶ－ＩｇＭ、献血员筛选及防止丙型肝炎传播。     

丙型肝炎患者外周血单个核细胞HCV免疫组化和电镜研究

目的：探讨丙型肝炎患者外周血单个核细胞（PBMCs）内丙型肝炎病毒抗原（HCVAg）表达和HCV颗粒感染状况及意义。方法：采用抗HCV核心区及NS3区单克隆抗体，对35例慢性丙肝患者PBMCs进行免疫组化检测和电镜观察。结果：慢性丙肝患者PBMCs的HCVAg阳性率为82.9%(29/35），其中82.8%（24/29）的病例电镜下发现了病毒颗粒，占全组病例的68.6%(24/35)。同时发现部分细胞超微结构呈现一系列凋亡性改变。结论：HCV可感染PBMCs并在其中复制，这可能是导致患者HCV持续感染的原因之一。     

慢性丙型肝炎患者血清HCVRNA水平与AST/ALT比例的关系

目的分析抗-HCV阳性患者血清水平HCVRNA与AST/ALT活性的关系;评价AST/ALT比值,HCVRNA水平在判断丙肝患者预后方面的价值。方法采用ELISA法检测抗-HCV,阳性标本分别用荧光PCR试剂盒测定HCVRNA;全自动生化分析仪检测AST/ALT。结果 78份抗-HCV阳性标本经PCR检测后有31份标本含有HCVRNA,HCVRNA拷贝量与AST/ALT比例呈正相关。结论动态监测AST/ALT比例和HCVRNA的水平,可用于丙型肝炎的预后估计。